In modern molecular biology and pharmacology, it has been found that a variety of factors are involved in producing a specific biological response. On the molecular level receptor-mediated cellular processes, and binding of effector molecules may result from combinations of hydrophobic, aromatic, charge transfer, salt bridging and hydrogen bonding interactions. For productive binding to occur it is vital that these forces and the groups which mediate them be displayed in appropriate conformations. A bioactive molecule may have agonist or antagonist activity, and may be a vital component of therapeutically useful compounds. Activity involves, inter alia, the several interactions mentioned of a biologically active molecule, a ligand, to a target molecule. An example is where a small ligand effectively blocks a larger ligand from binding to its target molecule, a natural biological receptor, e.g., to prevent the target from having an adverse effect on a tissue or organism. Conversely, a ligand may "turn on" a target molecule to engage in or initiate activity e.g., biochemical signal transduction.
These structure activity relationships are complex 3-D puzzles complicated by the in situ environment and the nature of the respective substituent groups of the ligand and target. The target and ligand molecules in fact are somewhat flexible, inter alia because of the manner in which the constituent atoms thereof bond to themselves and other molecules. For example, the flexure may be due in part to hydrogen bonding of different degrees of strength at different places along the molecules, and to the rotation around covalent bonds in the ligand molecule. Further, the isoelectric strength of the medium and the environment of the molecules plays a very important role. Steric hindrance by surfaces upon which small molecules are displayed may totally mask significant binding observable in solution. Display in proximity to surfaces, or within gel type polymers, will be similarly effected by the effective dielectric constant, changing the magnitude of all binding forces, and grossly affecting conformation, and therefore the "fit" between molecules.
Additionally, there are a wide range of degrees of affinity "fit" between ligand and target, and likewise between target and receptors, ranging from partial to complete fit. There is a concomitant range of effectiveness of a ligand to bind to target molecules, and thereby trigger a natural receptor, block, or ameliorate the adverse biological effects of the target on natural receptors, or inhibit the activity of an enzyme.
Another aspect of the problem is the vast numbers of potential candidate ligands considering the enormous number of molecular, isomeric and polymeric formulas. These numbers are increased by orders of magnitude when considering the variety of steric configurations and range of flexure. For example, considering the screening of hexapeptide ligands for biological blocking affinity of target molecules, there are some 64 million hexapeptides constructable from all the combinations of the 20 D-amino acids, an equal number for the 20 L-amino acids, and an even greater number for non-natural amino acids. Where the ligands are longer, the molecules have a greater chance of adopting a number of different conformations and thereby result in presentation of a number of different possible affinity combinations.
The two commonly used approaches rely either on solution interactions of the library ligand with a target, or supporting the ligand on a solid phase. The advantages of a supported library over solution interaction is the ability to rapidly identify ligands by content addressability, or to sequence the ligand or a surrogate tag for the ligand after identification. In contrast, deconvolution of soluble libraries only allows identification of concensus ligands, but not individual ligands.
Accordingly, there have been created various sized "libraries" of related organic molecules, i.e., a pre-selected set of ligand variations, on different types of solid supports. A variety of methods are known for producing such solid-phase supported libraries of organic molecules (ligands) as an aid in drug discovery. These include the Selectide one peptide per bead approach, the Affymax photolithographic approach, and the Arris PILOT addressable array approaches. The PILOT approach is the subject of co-pending Ser. No. 07/939,065, and the Selectide and Affymax approaches are summarized in the background thereof.
A major problem of potential library methods, is how to "display" the combinatorial ligand constituents, the concept of the term "display" including holding the ligand on or securing it to the substrate. Many ligand molecules have very low solubility in aqueous media, which is the usual solvent or carrier for target molecules flowed thereover during affinity screening. If the ligands are displayed on glass, silicon or polystyrene surfaces, for example, they can interact strongly with the surface on which they are displayed, introducing an artifact into the screening process. With the Affymax procedure, the bulk effect of the rigid silica matrix not only restricts the synthetic efficiency, but also introduces steric hindrance barriers to access by the target acceptor molecules.
Access to displayed ligands by target molecules is critical for success of the screening process. These target molecules are often large proteins or nucleotides which cannot easily diffuse into cross-linked gels.
It is particularly noteworthy that the Selectide process uses cross-linked polyacrylamide and polystyrene particles which permit reasonably efficient syntheses, but the cross-linking prevents access of biological target acceptors to any other than the surface molecules.
Accordingly, there is a need for a minimally sterically hindered system and method for the assembly and/or display of ligands for identification of binding constituents of target molecules that does not introduce unnatural steric artifacts or hindrances, yet is amenable to rapid, addressable screening techniques, the goal being highly sensitive biospecific ligand/target molecule interaction screening, purification, isolation, recovery and analysis.